The present invention relates to human collagen processing and autoimplant use, and more particularly to a chemically modified, crosslinkable, telopeptide-containing, naturally crosslinked, solubilized collagenous substance obtained directly from intact human tissue from a sole human donor, for implanting in various forms in the same said donor, and to the process for making such product.
In general, the collagens are ubiquitous proteins found throughout the animal kingdom. All known collagens are rod-like structures. Interstitial collagens are 3,000 .ANG. long and 15 .ANG. in diameter. The conformation and most of the properties of native collagen are determined by the triple helix domain which composes more than 95% of the molecule. This domain consists of three chains (alpha chains), each containing approximately 1,000 amino acids, wrapped in rope like fashion to form a tight, triple helix structure. The triple helix is wound in such a way that peptide bonds linking adjacent amino acids are buried within the interior of the molecule.
In native molecules the triple helix retains its resistance to attack by general proteases such as pepsin. Collagen molecules (tropocollagen) found in the extracellular matrices also contain short (e.g. about 16-25 peptide unit) non-helical extension peptides, called "telopeptides", at both the NH-- and COOH-- terminal ends of each alpha chain. These telopeptides are susceptible to proteolytic degradation and removal under conditions in which the triple helical body is left intact (as atelopeptide collagen).
Native collagen is generally present in connective tissue as telopeptide-containing tropocollagen molecules in side by side packed condition in the form of fibrils, with each longitudinal course composed of slightly longitudinally spaced apart molecules in end to end disposition, staggered longitudinally relative to the next successive laterally adjacent longitudinal course, thereby resulting in holes between facing end regions of successive molecules in a given longitudinal course and bounded by the staggered sides of the molecules in the parallel longitudinal courses laterally adjacent thereto.
These fibrils, e.g. of about 5 to 7 parallel courses packed together, in turn are arranged in bundles to form fibers which, along with the cells themselves, exist in the tissue in a ground substance of noncollagenous material as matrix. In bone, such holes in the staggered packing arrangement may contain mineral substances such as calcium phosphates.
In this native form, adjacent telopeptide-containing end moieties of given molecule in a fibril are crosslinked to helical regions of adjacent molecules. The helical or central regions of the polypeptide chains or strands of a given molecule are crosslinked to each other (intramolecular crosslinks) to form a triple helix. The telopeptide and helical regions of neighboring molecules are likewise crosslinked to strands of neighboring molecules (intermolecular crosslinks), thereby forming hydrogen crosslinked or bonded and covalently crosslinked or condensed insoluble collagen. Where few, if any, stabilized reducible, crosslinks are present, the molecules in the fibril are considered soluble, i.e. the collagen is solubilized in aqueous salts, acids and bases, leaving unsolubilized the highly stabilized, crosslinked insoluble collagen.
The most common type of collagen isolated from many adult connective tissues such as skin, bone, tendon, and cornea is type I collagen. Each type I molecule is composed of two alpha 1 (I) chains and one alpha 2 (I) chain. The entire molecule is abbreviated alpha 1 (I).sub.2 alpha 2 (I).
Collagen is probably the first biomaterial ever used by man for surgical purposes. Dried intestine, predominantly composed of collagen, was used by Egyptian surgeons as a surgical suture as far back as 3750 B.C.
Numerous properties of collagen favor its use as a biomaterial; see Biomaterials in Reconstructive Surgery, Ch. 11, Simpson, "Collagen as a biomaterial", pp. 109-117, The C. V. Mosby Co., 1983. It is absorbed at a rate that can be controlled by the degree of chemical treatment to which it is subjected. One can thus design collagen products which, on animal implantation, will be completely absorbed in a few-days or months. One can chemically treat animal source collagen so that it becomes essentially totally non-absorbable while still retaining its hydrophilic character and its good tissue response.
Collagen has a high order of tensile strength and low extensibility, and can be reconstituted into membranes, sheets, tubes, sponges, or continuous length fibers. As a membrane, is semi-permeable and a good support for cell growth. It has drug binding properties and is, for all practical purposes, immunologically inert.
The chemical and physical characteristics of collagen, its widespread distribution in many different tissues, and the ability to extract and purify and then reconstitute collagen into many physical forms would appear to make the natural polymer an ideal biomaterial. Many applications for collagen compositions have been suggested:
(A) solution form collagen applications: plasma expander, and drug delivery vehicle;
(B) gel form collagen applications: vitreous body additive, and cosmeticum;
(C) flour form collagen application: hemostatic agent;
(D) fiber form collagen applications: suture material, weaving of blood vessels, and valve prosthesis;
(E) film or membrane form collagen applications: corneal replacement, hemodialysis, artificial kidneys, wound dressing, hernia repair, and patches (aneurysm);
(F) sponge form collagen applications: wound dressing, bone-cartilage substitute, surgical tampon, and vaginal contraceptive; and
(G) tubing form collagen applications: vessel prosthesis, and reconstructive surgery of hollow organs.
However, until recently, the only clinically available collagen device was animal source suture material from intestines and from reconstituted collagen. Today there are at least two additional clinical devices composed of animal source collagen, to wit, hemostatic agents, and the Zyderm Collagen Implant (Collagen Corporation) or ZCI; see Grosh et al, J. Am. Acad. Dermatol., 13: 792-798, 1985.
Pertinent prior art describes methods of chemically modifying soluble collagen by reactions with either amine or carboxyl groups on the collagen molecule. These methods render the solubilized collagen soluble at physiological pH. Collagen is generally solubilized by treatment with acids, including organic acids such as acetic acid and citric acid, and inorganic acids such as hydrochloric acid, and especially by proteolyric enzyme treatment. The solubilized collagens contain few, if any, intermolecular crosslinks and remain soluble under acidic conditions and spontaneously form fibers at physiological pH.
Modification of either amine or carboxylic moieties changes the pK of the molecule. For example, by modification with succinic anhydride, the pK changes from 7.0 to 4.3. The succinylated collagen will remain soluble at pH 7.0 and will form fibers at pH 4.3.
These overall methods, however, generally removed the telopeptide groups.
SUMMARY OF THE INVENTION
It is among the objects of this invention to produce a chemically modified, crosslinkable, telopeptide-containing, naturally crosslinked, solubilized collagenous substance obtained directly from intact human tissue from a sole donor, for altering the condition of in situ tissue of the same donor, e.g. for augmenting soft tissue, by autoimplantation.
Briefly, this invention concerns the processing of collagens from a biopsy or other specimen of human skin or other human tissue (e.g. skin or bone for Type I fibrous collagen, or cartilage for Type II collagen), for use as a biological autoimplant in the same tissue donor alone.
Such autoimplants contemplate two major categories, i.e. intradermal implants to augment soft connective tissue or correct skin defects such as wrinkles and scars; and ophthalmic implants, e.g. intralamellar, corneal overlay coating or reshaping, vitreous, and other implants, in refractive surgery to correct refractive errors of vision, change corneal curvature, replace vitreous humor, and the like; as well as other categories of implants such as those used in other surgical procedures where there is a need to replace, augment or otherwise change the condition of connective tissue, e.g. in the form of matrix material for skin grafts, matrix substances or components for cell seeding and grafting, material matrix for tissue "putty" or filler, and the like.
This invention also concerns novel processing techniques for extraction from intact human tissue of insoluble, naturally crosslinked, native, telopeptide-containing collagen by reacting such collagen, obtained from the donor patient alone, with chemical reagents that render the insoluble collagen more soluble in physiological aqueous solutions, significantly without acidic or alkaline hydrolysis or enzymatic degradation, and such that the extracted or solubilized telopeptide-containing, naturally crosslinked, collagens can be further purified and then chemically or physically treated to provide fibrous structures, flour like particles, gels, sponges, clear and colorless solutions, or suspensions, or the like for autoimplanting in the same human donor.
In intact human tissue, the telopeptide-containing triple helix collagen units of the staggered packed array of tropocollagen molecules of the fibrils, are in highly crosslinked, insoluble condition. The helical or central regions are high in glycine, proline and hydroxyproline amino acid residues, and the telopeptide or end appendage regions contain aromatic residues (tyrosine) and do not exhibit the glycine-X-Y-triplet found in the helical region.
The individual helical chains or strands of the triple helix molecules are arranged in side by side intramolecularly. and/or intermolecularly crosslinked disposition along the corresponding collagen polypeptide backbone, such that the terminal amino group-containing site of each given strand is linked to its adjacent non-helical telopeptide end moiety, and the terminal carboxylic acid group-containing site of the same strand is linked to its adjacent non-helical telopeptide end moiety. The nonhelical regions are crosslinked, intramolecularly, with helical regions of adjacent molecules.
Heretofore, in normal processing to extract the collagen by solubilization, conditions were used which resulted in the severing of the helical strands from one another and/or the severing of the strands from their telopeptide units to form individual triple helix collagen strand subunits or atelopeptides. This normally rendered the resulting atelopeptide or solubilized collagen soluble at acidic pH and insoluble at neutral pH.
By way of the invention, the extraction and recovery of the collagen from the human tissue is carried out essentially without severing the triple helical strands from each other, or the telopeptide end moieties from the opposite-ends of the helical regions of the individual strands. Thus, the original intact linking of the individual units along the polypeptide backbone, and the original intact natural crosslinking between adjacent helical strands and between adjacent non-helical telopeptide end units, are essentially preserved. Instead, the intact collagen is chemically modified to solubilize it at neutral or basic pH, and render it insoluble at acidic pH.
Unlike previously used acid soluble and enzyme digested forms of extracted and chemically modified atelopeptide collagen products, the telopeptide-containing, naturally crosslinked, collagen product of this invention is believed to be more compatible with the tissue environment of the same human donor, and more resistant to degradation, absorption, rejection, or other attack by in situ constituents of such donor, possibly because it is desirably made free from noncollagenous protein contaminates, and preferably also from lipid constituents, but more particularly because it preserves the telopeptide moleties and the natural crosslinks and chemically provides additional crosslinking sites.
No antigenic potential need be feared since the human tissue processing contemplated by this invention involves only autologous tissue, i.e. obtained from the very same person in whom the product is reimplanted, as opposed to heterologous tissue, i.e. obtained from another person than the one in whom the product is transplanted.
Hence, per this invention, due to the autologous nature of the human tissue, no antibody response or rejection is to be expected, whereas due to the contemplated chemical modifying and crosslinking of the product, the autoimplanted product will serve as a relatively more permanent implant material than previously known products.
Nevertheless, based on this specific autologous tissue distinction, over the known heterologous tissue use, this invention also broadly permits altering the condition of in situ tissue of a human donor by autoimplantation, using an autoimplantable or reimplantable, processed collagenous substance derived from intact tissue of the very same human donor alone, regardless of the means or process used to extract and chemically modify the tissue, and whether the processing is such that the completely or partially solubilized collagen still contains telopeptide moleties, as is preferred, or results in the less preferred formation of completely or partially solubilized atelopeptide collagen as in the past.
This is because a salient independent feature of this invention concerns the concept of autoimplantation of a collagenous substance product in a human donor which has been derived from intact tissue of that same donor alone, thus avoiding potential problems associated with antigenicity, rejection and the like of heterologous tissue transplants.
This invention thus provides forms of processed human tissue derived collagen serving as a long-term, practical and relatively safe autoimplant product, e.g. permitting its production almost contemporaneously with its use in a given surgical procedure as a corneal, skin, coating, interconnecting layer, or the like implant at a surgical site. Of course, all such procedures are effected under sterile, antiseptic conditions using sterile materials.